Biomedical subjects

A S Verkman

Publications and source records attributed to A S Verkman.

At least 19 recordsLinked to original sources

PubMed2007

Urearetics: a small molecule screen yields nanomolar potency inhibitors of urea transporter UT-B.

Functional studies in knockout mice indicate a critical role for urea transporters (UTs) in the urinary concentrating mechanism and in renal urea clearance. However, potent and specific urea transport blockers have not been available. Here, we used high-throughput screening to discover high-affinity, small molecule inhibitors of the UT-B urea transporter. A collection of 50,000 diverse, drug-like compounds was screened using a human erythrocyte lysis assay based on UT-B-facilitated acetamide transport. Primary screening yielded approximately 30 UT-B inhibitors belonging to the phenylsulfoxyoxazole, benzenesulfonanilide, phthalazinamine, and aminobenzimidazole chemical classes. Screening of approximately 700 structurally similar analogs gave many active compounds, the most potent of which inhibited UT-B urea transport with an EC50 of approximately 10 nM, and approximately 100% inhibition at higher concentrations. Phenylsulfoxyoxazoles and phthalazinamines also blocked rodent UT-B and had good UT-B vs. UT-A specificity. The UT-B inhibitors did not reduce aquaporin-1 (AQP1)-facilitated water transport. In AQP1-null erythrocytes, "chemical UT-B knockout" by UT-B inhibitors reduced by approximately 3-fold UT-B-mediated water transport, supporting an aqueous pore pathway through UT-B. UT-B inhibitors represent a new class of diuretics, "urearetics," which are predicted to increase renal water and solute clearance in water-retaining states.

Animals↗

PubMed2006

Greatly impaired migration of implanted aquaporin-4-deficient astroglial cells in mouse brain toward a site of injury.

We reported previously that astroglia cultured from aquaporin-4-deficient (AQP4-/-) mice migrate more slowly in vitro than those from wild-type (AQP4+/+) mice (J. Cell Sci. 2005;118, 5691-5698). Here, we investigate the migration of fluorescently labeled AQP4+/+ and AQP4-/- astroglia after implantation into mouse brains in which directional movement was stimulated by a planar stab wound 3 mm away from the axis of the injection needle. Two days after cell injection we determined the location, elongation ratio, and orientation of labeled cells. Migration of AQP4+/+ but not AQP4-/- cells toward the stab was greater than away from the stab. AQP4+/+ astroglia moved on average 1.5 mm toward the stab compared with 0.6 mm for AQP4-/- cells. More than 25% of the migrating AQP4+/+ cells but <3% of AQP4-/- cells appeared elongated (axial ratio>2.5). In transwell assays, AQP4+/+ astroglia migrated faster than AQP4-/- cells in a manner dependent on pore size. At 8 h, approximately 50% of AQP4+/+ cells migrated through 8-microm diameter pores, whereas equivalent migration of AQP4-/- cells was found for 12-microm diameter pores. These results provide in vivo evidence for AQP4-dependent astroglial migration and suggest that modulation of AQP4 expression or function might alter glial scarring.

Animals↗

PubMed2006

Comparative efficacy of HgCl2 with candidate aquaporin-1 inhibitors DMSO, gold, TEA+ and acetazolamide.

Aquaporin-1 (AQP1) inhibitors are predicted to have multiple clinical applications. Hg(++) is a non-specific and toxic AQP1 blocker. We compared compounds with reported AQP1 inhibition activity, including DMSO, Au(+++), Ag(+), tetraethylammonium and acetazolamide. Water permeability was measured by stopped-flow light scattering in erythrocytes and volume marker dilution in epithelial cells. Au(+++) inhibited AQP1 with IC50 approximately 14 microM, similar to 10 microM for Hg(++). DMSO slowed osmotic equilibration; however, the apparent inhibition was due to 'osmotic clamp' rather than AQP1 inhibition. Neither tetraethylammonium nor acetazolamide (to 10 mM) inhibited AQP1. Our data indicate the need to identify new AQP1 inhibitors.

Acetazolamide↗

PubMed2006

Millisecond association kinetics of K+ with triazacryptand-based K+ indicators measured by fluorescence correlation spectroscopy.

We recently introduced a water-soluble, long-wavelength K(+)-sensing indicator, TAC-Red, consisting of a triazacryptand K(+)-selective ionophore coupled to a xanthylium chromophore (Nat. Methods 2005, 2, 825-827). Stopped-flow kinetic analysis indicated that in response to changes in K(+) concentration TAC-Red fluorescence enhancement occurs in milliseconds or less. Here, we use fluorescence correlation spectroscopy to quantify the binding kinetics of K(+) with TAC-Red and a new, longer-wavelength sensor, TAC-Crimson. Autocorrelation functions, G(tau), were similar at 0 and high (150 mM) K(+) concentrations, with the appearance of a prominent kinetic process with a correlation time in the millisecond range for K(+) concentrations between approximately 20 and 60 mM. Control experiments with increased illumination volume and solution viscosity indicated that the millisecond component represented K(+)/TAC-Red association. K(+)-dependent G(tau) data, modeled using a global regression to a binding/diffusion model, gave association and dissociation rate constants of 0.0020 +/- 0.0003 mM(-1) ms(-1) and 0.12 +/- 0.02 ms(-1), respectively, for TAC-Red. Similar results were obtained for TAC-Crimson. The rapid K(+) binding kinetics with triazacryptand-based sensors support their utility for measuring changes in K(+) concentrations during rapid neural signaling and ion channel gating.

Cations↗

PubMed2006

Tracking of quantum dot-labeled CFTR shows near immobilization by C-terminal PDZ interactions.

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100-200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.

Actins↗

PubMed2006

Small molecules with antimicrobial activity against E. coli and P. aeruginosa identified by high-throughput screening.

BACKGROUND AND PURPOSE: New antimicrobials are needed because of the emergence of organisms that are resistant to available antimicrobials. The purpose of this study was to evaluate a high-throughput screening approach to identify antibacterials against two common disease-causing bacteria, and to determine the frequency, novelty, and potency of compounds with antibacterial activity. EXPERIMENTAL APPROACH: A high-throughput, turbidometric assay of bacterial growth in a 96-well plate format was used to screen a diverse collection of 150,000 small molecules for antibacterial activity against E. coli and P. aeruginosa. The statistical Z'-factor for the assay was > or = 0.7. KEY RESULTS: Screening for inhibition of E. coli growth gave a 'hit' rate (> 60% inhibition at 12.5 microM) of 0.025%, which was more than 5-fold reduced for P. aeruginosa. The most potent antibacterials (EC50 < 0.5 microM) were of the nitrofuran class followed by naphthalimide, salicylanilide, bipyridinium and quinoazolinediamine chemical classes. Screening of > 250 analogs of the most potent antibacterial classes established structure-activity data sets. CONCLUSIONS AND IMPLICATIONS: Our results validate and demonstrate the utility of a growth-based phenotype screen for rapid identification of small-molecule antibacterials. The favourable efficacy and structure-activity data for several of the antibacterial classes suggests their potential development for clinical use.

Anti-Bacterial Agents↗

PubMed2006

Aquaporins and CFTR in ocular epithelial fluid transport.

Aquaporins (AQPs) and the cystic fibrosis transmembrane conductance regulator (CFTR) provide the molecular routes for transport of water and chloride, respectively, through many epithelial tissues. In ocular epithelia, fluid transport generally involves secondary active chloride transport, which creates the osmotic gradient to drive transepithelial water transport. This review is focused on the role of AQPs and CFTR in water and ion transport across corneal/conjunctival epithelia, corneal endothelium, ciliary epithelium, and retinal pigment epithelium. The potential relevance of water and chloride transport to common disorders of ocular fluid balance is also considered. Recent data suggest AQPs and CFTR as attractive targets for drug development for therapy of keratoconjunctivitis sicca, recurrent corneal erosions, corneal edema, glaucoma, retinal detachment, and retinal ischemia.

Animals↗

PubMed2006

Hypertonic saline therapy in cystic fibrosis: Evidence against the proposed mechanism involving aquaporins.

Recent data indicate the clinical benefit of nebulized hypertonic saline in cystic fibrosis lung disease, with a proposed mechanism involving sustained increase in airway surface liquid volume. To account for the paradoxical observation that amiloride suppresses the beneficial effect of hypertonic saline, it has been previously concluded (Donaldson, S. H., Bennett, W. D., Zeman, K. L., Knowles, M. R., Tarran, R., and Boucher, R. C. (2006) N. Engl. J. Med. 354, 241-250) that amiloride-inhibitable aquaporin (AQP) water channels in airway epithelia modulate airway surface liquid volume. Here, we have characterized water permeability and amiloride effects in well differentiated, primary cultures of human airway epithelial cells, stably transfected Fisher rat thyroid epithelial cells expressing individual airway/lung AQPs, and perfused mouse lung. We found high transepithelial water permeability (P(f), 54 +/- 5 microm/s) in airway epithelial cells that was weakly temperature-dependent and inhibited by >90% by reduced pH in the basal membrane-facing solution. Reverse transcription-PCR and immunofluorescence suggested the involvement of AQPs 3, 4, and 5 in high airway water permeability. Experiments using several sensitive measurement methods indicated that amiloride does not inhibit water permeability in non-cystic fibrosis (non-CF) or CF airway epithelia, AQP-transfected Fisher rat thyroid cells, or intact lung. Our data provide evidence against the mechanism proposed by Donaldson et al. to account for the effects of amiloride and hypertonic saline in CF lung disease, indicating the need to identify alternate mechanisms.

Amiloride↗

PubMed2006

Increased migration and metastatic potential of tumor cells expressing aquaporin water channels.

Aquaporin (AQP) water channels are expressed in high-grade tumor cells of different tissue origins. Based on the involvement of AQPs in angiogenesis and cell migration, we tested whether AQP expression in tumor cells might enhance their migration and metastatic potential. Transfection of B16F10 and 4T1 tumor cells with AQP1 did not affect their appearance, size, growth, or substrate adherence but increased their plasma membrane osmotic water permeability by 5- to 10-fold. In vitro analysis of cell migration by transwell assay, wound healing and video microscopy showed a 2- to 3-fold accelerated migration of the AQP1-expressing tumor cells compared to control cells. In mice, AQP1 expression increased tumor cell extravasation by >1.5-fold as quantified by counting tumor cells in lung at 6 h after tail vein injection of a mixture of fluorescently tagged AQP1-expressing and control tumor cells. AQP1 expression also increased by 3-fold the number of lung metastases 14 days after tail vein injection of tumor cells, with alveolar wall infiltration seen with AQP1-expressing tumor cells. Our results provide evidence for AQP-facilitated tumor cell migration and spread, suggesting a novel function for AQP expression in high-grade tumors. AQP inhibition may thus reduce the metastatic potential of some tumors.

Animals↗

PubMed2006

Evidence against functionally significant aquaporin expression in mitochondria.

Recent reports suggest the expression of aquaporin (AQP)-type water channels in mitochondria from liver (AQP8) (Calamita, G., Ferri, D., Gena, P., Liquori, G. E., Cavalier, A., Thomas, D., and Svelto, M. (2005) J. Biol. Chem. 280, 17149-17153) and brain (AQP9) (Amiry-Moghaddam, M., Lindland, H., Zelenin, S., Roberg, B. A., Gundersen, B. B., Petersen, P., Rinvik, E., Torgner, I. A., and Ottersen, O. P. (2005) FASEB J. 19, 1459-1467), where they were speculated to be involved in metabolism, apoptosis, and Parkinson disease. Here, we systematically examined the functional consequence of AQP expression in mitochondria by measurement of water and glycerol permeabilities in mitochondrial membrane preparations from rat brain, liver, and kidney and from wild-type versus knock-out mice deficient in AQPs -1, -4, or -8. Osmotic water permeability, measured by stopped-flow light scattering, was similar in all mitochondrial preparations, with a permeability coefficient P(f) approximately 0.009 cm/s. Glycerol permeability was also similar ( approximately 5 x 10(-6) cm/s) in the various preparations. HgCl(2) slowed osmotic equilibration comparably in mitochondria from wild-type and AQP-deficient mice, although the slowing was explained by altered mitochondrial size rather than reduced P(f). Immunoblot analysis of mouse liver mitochondria failed to detect AQP8 expression, with liver homogenates from wild-type/AQP8 null mice as positive/negative controls. Our results provide evidence against functionally significant AQP expression in mitochondria, which is consistent with the high mitochondrial surface-to-volume ratio producing millisecond osmotic equilibration, even when intrinsic membrane water permeability is not high.

Animals↗

PubMed2006

Evidence from knockout mice against physiologically significant aquaporin 8-facilitated ammonia transport.

Aquaporin (AQP)8-facilitated transport of NH(3) has been suggested recently by increased NH(3) permeability in Xenopus oocytes and yeast expressing human or rat AQP8. We tested the proposed roles of AQP8-facilitated NH(3) transport in mammalian physiology by comparative phenotype studies in wild-type vs. AQP8-null mice. AQP8-facilitated NH(3) transport was confirmed in mammalian cell cultures expressing rat or mouse AQP8, in which the fluorescence of a pH-sensing yellow fluorescent protein was measured in response to ammonia (NH(3)/NH(4)(+)) gradients. Relative AQP8 single-channel NH(3)-to-water permeability was approximately 0.03. AQP8-facilitated NH(3) and water permeability in a native tissue was confirmed in membrane vesicles isolated from testes of wild-type vs. AQP8-null mice, in which BCECF was used as an intravesicular pH indicator. A series of in vivo studies were done in mice, including 1) serum ammonia measurements before and after ammonia infusion, 2) renal ammonia clearance, 3) colonic ammonia absorption, and 4) liver ammonia accumulation and renal ammonia excretion after acute and chronic ammonia loading. Except for a small reduction in hepatic ammonia accumulation and increase in ammonia excretion in AQP8-null mice loaded with large amounts of ammonia, there were no significant differences in wild-type vs. AQP8-null mice. Our results support the conclusion that AQP8 can facilitate NH(3) transport but provide evidence against physiologically significant AQP8-facilitated NH(3) transport in mice.

Ammonia↗

PubMed2006

Cardiac aquaporin expression in humans, rats, and mice.

Water accumulation in the heart is important in ischemia-reperfusion injury and operations performed by using cardiopulmonary bypass, with cardiac dysfunction associated with myocardial edema being the principal determinant of clinical outcome. As an initial step in determining the role of aquaporin (AQP) water channels in myocardial edema, we have assessed the myocardial expression of AQPs in humans, rats, and mice. RT-PCR revealed expression of AQP-1, -4, -6, -7, -8, and -11 transcripts in the mouse heart. AQP-1, -6, -7, and -11 mRNAs were found in the rat heart as well as low levels of AQP-4 and -9. Human hearts contained AQP-1, -3, -4, -5, -7, -9, -10, and -11 mRNAs. AQP-1 protein expression was confirmed by Western blot analysis in all three species. AQP-4 protein was detected in the mouse heart but not in the rat or human heart. To determine the potential functional consequences of myocardial AQP expression, water permeability was measured in plasma membrane vesicles from myocardial cells of wild-type versus various AQP knockout mice. Water permeability was reduced by AQP-1 knockout but not by AQP-4 or AQP-8 knockout. With the use of a model of isolated rat heart perfusion, it was found that osmotic and ischemic stresses are not associated with changes in AQP-1 or AQP-4 expression. These studies support a possible functional role of AQP-1 in myocardium but indicate that early adaptations to osmotic and ischemic stress do not involve transcriptional or posttranslational AQP-1 regulation.

Animals↗

PubMed2006

Role of RhAG and AQP1 in NH3 and CO2 gas transport in red cell ghosts: a stopped-flow analysis.

To clarify the potential role Rh/RhAG and AQP1 proteins in erythrocyte gas transport, NH3 and CO2 transport was measured in erythrocyte ghost membrane vesicles from rare human variants (Rh(null), CO(null),) and knockout mice (homozygous AQP1-/-, Rh-/- and Rhag-/-) exhibiting well-characterized protein defects. Transport was measured from intracellular pH (pHi) changes in a stopped-flow fluorimeter. NH3 transport was measured in chloride-free conditions with ghosts exposed to 20 mM inwardly directed gradients of gluconate salts of ammonium, hydrazine and methylammonium at 15 degrees C. Alkalinization rates of control samples were 6.5+/-0.3, 4.03+/-0.17, 0.95+/-0.08 s(-1) for each solute, respectively, but were significantly reduced for Rh(null) and CO(null) samples that are deficient in RhAG and AQP1 proteins, respectively. Alkalinization rates of Rh(null) ghosts were about 60%, 83% and 94% lower than that in control ghosts, respectively, for each solute. In CO(null) ghosts, the lack of AQP1 resulted in about 30% reduction of the alkalinization rates as compared to controls, but the transport selectivity of RhAG for the three solutes was preserved. Similar observations were made with ghosts from KO mice Rhag-/- and AQP1-/-. These results confirm the major contribution of RhAG/Rhag in the NH3 conductance of erythrocytes and suggest that the reduction of transport rates in the absence of AQP1 would be better explained by a direct or indirect effect on RhAG/Rhag-mediated transport. When ghosts were preloaded with carbonic anhydrase and exposed to a 25 mM CO2/HCO3- gradient at 6 degrees C, an extremely rapid kinetics of acidification corresponding to CO2 influx was observed. The rate constants were not significantly different between controls and human variants (125+/-6 s(-1)), or between wild-type and KO mice, suggesting no major role of RhAG or AQP1 in CO2 transport, at least in our experimental conditions.

Ammonia↗

PubMed2006

Accelerated progression of kaolin-induced hydrocephalus in aquaporin-4-deficient mice.

Hydrocephalus is caused by an imbalance in cerebrospinal fluid (CSF) production and absorption, resulting in excess ventricular fluid accumulation and neurologic impairment. Current therapy for hydrocephalus involves surgical diversion of excess ventricular fluid. The water-transporting protein aquaporin-4 (AQP4) is expressed at the brain-CSF and blood-brain barriers. Here, we provide evidence for AQP4-facilitated CSF absorption in hydrocephalus by a transparenchymal pathway into the cerebral vasculature. A mouse model of obstructive hydrocephalus was created by injecting kaolin (2.5 mg/mouse) into the cisterna magna. Intracranial pressure (ICP) was approximately 5 mm Hg and ventricular size <0.3 mm(3) in control mice. Lateral ventricle volume increased to 3.7+/-0.5 and 5.1+/-0.5 mm(3) in AQP4 null mice at 3 and 5 days after injection, respectively, significantly greater than 2.6+/-0.3 and 3.5+/-0.5 mm(3) in wildtype mice (P<0.005). The corresponding ICP was 22+/-2 mm Hg at 3 days in AQP4 null mice, significantly greater than 14+/-1 mm Hg in wildtype mice (P<0.005). Brain parenchymal water content increased by 2% to 3% by 3 days, corresponding to approximately 50 muL of fluid, indicating backflow of CSF from the ventricle into the parenchymal extracellular space. A multi-compartment model of hydrocephalus based on experimental data from wildtype mice accurately reproduced the greater severity of hydrocephalus in AQP4 null mice, and predicted a much reduced severity if AQP4 expression/function were increased. Our results indicate a significant role for AQP4-mediated transparenchymal CSF absorption in hydrocephalus and provide a rational basis for evaluation of AQP4 induction as a nonsurgical therapy for hydrocephalus.

Animals↗

PubMed2006

Three distinct roles of aquaporin-4 in brain function revealed by knockout mice.

Aquaporin-4 (AQP4) is expressed in astrocytes throughout the central nervous system, particularly at the blood-brain and brain-cerebrospinal fluid barriers. Phenotype analysis of transgenic mice lacking AQP4 has provided compelling evidence for involvement of AQP4 in cerebral water balance, astrocyte migration, and neural signal transduction. AQP4-null mice have reduced brain swelling and improved neurological outcome in models of (cellular) cytotoxic cerebral edema including water intoxication, focal cerebral ischemia, and bacterial meningitis. However, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema including cortical freeze-injury, brain tumor, brain abscess and hydrocephalus, probably due to impaired AQP4-dependent brain water clearance. AQP4 deficiency or knock-down slows astrocyte migration in response to a chemotactic stimulus in vitro, and AQP4 deletion impairs glial scar progression following injury in vivo. AQP4-null mice also manifest reduced sound- and light-evoked potentials, and increased threshold and prolonged duration of induced seizures. Impaired K+ reuptake by astrocytes in AQP4 deficiency may account for the neural signal transduction phenotype. Based on these findings, we propose modulation of AQP4 expression or function as a novel therapeutic strategy for a variety of cerebral disorders including stroke, tumor, infection, hydrocephalus, epilepsy, and traumatic brain injury.

Action Potentials↗

PubMed2006

Fluorescence correlation spectroscopy simulations of photophysical phenomena and molecular interactions: a molecular dynamics/monte carlo approach.

Fluorescence correlation spectroscopy (FCS) is being applied increasingly to study diffusion and interactions of fluorescently labeled macromolecules in complex biological systems. Fluctuations in detected fluorescence, deltaF(t), are expressed as time-correlation functions, G(tau), and photon-count histograms, P(k;DeltaT). Here, we developed a generalized simulation approach to compute G(tau) and P(k;DeltaT) for complex systems with arbitrary geometry, photophysics, diffusion, and macromolecular interactions. G(tau) and P(k;DeltaT) were computed from deltaF(t) generated by a Brownian dynamics simulation of single-molecule trajectories followed by a Monte Carlo simulation of fluorophore excitation and detection statistics. Simulations were validated by comparing analytical and simulated G(tau) and P(k;DeltaT) for diffusion of noninteracting fluorophores in a three-dimensional Gaussian excitation and detection volume. Inclusion of photobleaching and triplet-state relaxation produced significant changes in G(tau) and P(k;DeltaT). Simulations of macromolecular interactions and complex diffusion were done, including transient fluorophore binding to an immobile matrix, cross-correlation analysis of interacting fluorophores, and anomalous sub- and superdiffusion. The computational method developed here is generally applicable for simulating FCS measurements on systems complicated by fluorophore interactions or molecular crowding, and experimental protocols for which G(tau) and P(k;DeltaT) cannot be computed analytically.

Algorithms↗

PubMed2006

Impaired pain sensation in mice lacking Aquaporin-1 water channels.

Aquaporin-1 (AQP1), a membrane water channel, is expressed in choroid plexus where it contributes to cerebrospinal fluid production. Here, we show that AQP1 is also expressed in the dorsal horn of the spinal cord and the trigeminal nucleus caudalis, regions that process pain information. Within the dorsal root and trigeminal sensory ganglia, AQP1 is concentrated in small diameter cell bodies, most of which give rise to unmyelinated C-fibers. To study the role of AQP1 in pain signaling, we compared acute pain responses in wild-type mice and in mice lacking AQP1. AQP1(-/-) mice had reduced responsiveness to thermal and capsaicin chemical stimuli, but not to mechanical stimuli or formalin. These results provide evidence for AQP1 expression in nociceptive neurons and suggest that AQP1 may play a role in pain signal transduction.

Animals↗

PubMed2006

Mouse model of inducible nephrogenic diabetes insipidus produced by floxed aquaporin-2 gene deletion.

Transgenic mouse models of defective urinary concentrating ability produced by deletion of various membrane transport or receptor proteins, including aquaporin-2 (AQP2), are associated with neonatal mortality from polyuria. Here, we report an inducible mouse model of AQP2 gene deletion with severe polyuria in adult mice. LoxP sequences were inserted into introns 1 and 2 in the mouse AQP2 gene by homologous recombination in embryonic stem cells. Mating of germ-line AQP2-loxP mice with tamoxifen-inducible Cre-expressing mice produced offspring with inducible homozygous Cre-AQP2-loxP, which had a normal phenotype. Tamoxifen injections over 10 days resulted in AQP2 gene excision, with undetectable full-length AQP2 transcript in kidney and a >95% reduction in immunoreactive AQP2 protein. Urine osmolality decreased from approximately 2,000 to <500 mosmol/kgH(2)O after 4-5 days, with urine output increasing from 2 to 25 ml/day. Urine osmolality did not increase after water deprivation. Interestingly, AQP3 protein expression in the collecting duct was increased by about fivefold after AQP2 gene excision. Mild renal damage was seen after 6 wk of polyuria, with collecting duct dilatation, yet normal creatinine clearance and serum chemistries. These results establish the first adult model of nephrogenic diabetes insipidus (NDI) caused by AQP2 deficiency, with daily urine output comparable to body weight, although remarkable preservation of renal function compared with non-inducible NDI models.

Animals↗